Vaccine comprising protein nmb0964 from neisseria meningitidis

ABSTRACT

The present invention relates to immunogenic compositions comprising neisserial blebs with upregulated levels of the NMB0964 antigens such that bactericidal antibodies are generated against said antigen. It has been found for the first time that this antigen&#39;s expression is zinc regulated and therefore methods are provided to upregulated expression through removal of the zinc repression mechanism of the cell or promoter, or through removal of zinc from the culture medium.

This application is filed pursuant to 35 U.S.C. §121 as a divisionalapplication of U.S. Ser. No. 13/062,319 filed Mar. 4, 2011, a nationalphase entry of International Patent Application Serial No.PCT/EP2009/052689 filed Mar. 6, 2009, which claims priority toApplication No. GB 0816447.7 filed Sep. 8, 2008, the contents of whichare incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to immunogenic compositions for the prevention ofdiseases caused by Neisseria bacteria, in particular Neisseriameningitidis.

BACKGROUND OF THE INVENTION

Neisserial strains of bacteria are the causative agents for a number ofhuman pathologies, against which there is a need for effective vaccinesto be developed. In particular Neisseria gonorrhoeae and Neisseriameningitidis cause pathologies which could be treated by vaccination.

Neisseria gonorrhoeae is the etiologic agent of gonorrhea, one of themost frequently reported sexually transmitted diseases in the world withan estimated annual incidence of 62 million cases (Gerbase et al 1998Lancet 351; (Suppl 3) 2-4). The clinical manifestations of gonorrheainclude inflammation of the mucus membranes of the urogenital tract,throat or rectum and neonatal eye infections. Ascending gonococcalinfections in women can lead to infertility, ectopic pregnancy, chronicpelvic inflammatory disease and tubo-ovarian abscess formation.Septicemia, arthritis, endocarditis and menigitis are associated withcomplicated gonorrhea.

The high number of gonococcal strains with resistance to antibioticscontributes to increased morbidity and complications associated withgonorrhea. An attractive alternative to treatment of gonorrhea withantibiotics would be its prevention using vaccination. No vaccinecurrently exists for N. gonorrhoeae infections.

Neisseria meningitidis is an important pathogen, particularly inchildren and young adults. Septicemia and meningitis are the mostlife-threatening forms of invasive meningococcal disease (IMD). Thisdisease has become a worldwide health problem because of its highmorbidity and mortality.

Thirteen N. meningitidis serogroups have been identified based onantigenic differences in the capsular polysaccharides, the most commonbeing A, B and C which are responsible for 90% of disease worldwide.Serogroup B is the most common cause of meningococcal disease in Europe,USA and several countries in Latin America.

Vaccines based on the capsular polysaccharide of serogroups A, C, W andY have been developed and have been shown to control outbreaks ofmeningococcal disease (Peltola et al 1985 Pediatrics 76; 91-96). Howeverserogroup B is poorly immunogenic and induces only a transient antibodyresponse of a predominantly IgM isotype (Ala'Aldeen D and Cartwright K1996, J. Infect. 33; 153-157). There is therefore no broadly effectivevaccine currently available against the serogroup B meningococcus whichis responsible for the majority of disease in most temperate countries.This is particularly problematic since the incidence of serotype Bdisease is increasing in Europe, Australia and America, mostly inchildren under 5. The development of a vaccine against serogroup Bmeningococcus presents particular difficulties because thepolysaccharide capsule is poorly immunogenic owing to its immunologicsimilarity to human neural cell adhesion molecule. Strategies forvaccine production have therefore concentrated on the surface exposedstructures of the meningococcal outer membrane but have been hampered bythe marked variation in these antigens among strains.

Further developments have led to the introduction of vaccines made up ofouter membrane vesicles which will contain a number of proteins thatmake up the normal content of the bacterial membrane. One of these isthe VA-MENGOC-BC Cuban vaccine against N. meningitidis serogroups B andC (Rodriguez et al 1999 Mem Inst. Oswaldo Cruz, Rio de Janeiro 94;433-440). This vaccine was designed to combat an invasive meningococcaldisease outbreak in Cuba which had not been eliminated by a vaccinationprogramme using a capsular polysaccharide AC vaccine. The prevailingserogroups were B and C and the VA-MENGOC-BC vaccine was successful atcontrolling the outbreak with an estimated vaccine efficiency of 83%against serogroup B strains of N. meningitidis (Sierra et al 1990 InNeisseria, Walter Gruyter, Berlin, M. Achtman et al (eds) p 129-134,Sierra et al 1991, NIPH Ann 14; 195-210). This vaccine was effectiveagainst a specific outbreak, however the immune response elicited wouldnot protect against other strains of N. meningitidis.

Subsequent efficacy studies conducted in Latin America during epidemicscaused by homologous and heterologous serogroup B meningococcal strainshave shown some efficacy in older children and adults but itseffectiveness was significantly lower in younger children who are atgreatest risk of infection (Milagres et al 1994, Infect. Immun. 62;4419-4424). It is questionable how effective such a vaccine would be incountries with multistrain endemic disease such as the UK. Studies ofimmunogenicity against heterologous strains have demonstrated onlylimited cross-reactive serum bactericidal activity, especially ininfants (Tappero et al 1999, JAMA 281; 1520-1527).

A second outer membrane vesicle vaccine was developed in Norway using aserotype B isolate typical of those prevalent in Scandinavia (Fredriksenet al 1991, NIPH Ann, 14; 67-80). This vaccine was tested in clinicaltrials and found to have a protective efficacy after 29 months of 57%(Bjune et al 1991, Lancet, 338; 1093-1096).

There are diverse problems with the anti-meningococcal vaccinescurrently available. The protein based outer membrane vaccines tend tobe specific and effective against only a few strains. The polysaccharidevaccines are also suboptimal since they tend to elicit poor and shortimmune responses, particularly against serogroup B (Lepow et al 1986;Peltola 1998, Pediatrics 76; 91-96).

Neisseria infections represent a considerable health care problem forwhich no vaccines are available in the case of N. gonorrhoeae orvaccines with limitations on their efficacy and ability to protectagainst heterologous strains are available in the case of N.meningitidis. Clearly there is a need to develop superior vaccinesagainst Neisserial infections that will improve on the efficacy ofcurrently available vaccines and allow for protection against a widerrange of strains.

SUMMARY OF THE INVENTION

The present inventors have found that the Neisserial antigen NMB0964(NMB numbers refer to Neisseria meningitidis group B genome sequencesavailable from www.neisseria.org) [known as NMA1161 in the Neisseriameningitidis group A genome of strain Z2491, and as BASB082 in WO00/55327, and as ZnuD] is a conserved antigen throughout neisseria andcan induce bactericidal antibodies against a range of neisserialstrains. The inventors have found this antigen functions as a Zn²⁺receptor in the bacterium, and its expression is regulated by the levelof Zn²⁺ in the medium.

The present invention generally provides methods and compositions foreliciting an immune response against Neisseria spp. bacteria in asubject, particularly against a Neisseria meningitidis serogroup Bstrain.

In one aspect the present invention provides an immunogenic compositioncomprising: isolated outer membrane vesicles prepared from a Neisseriaspecies bacterium, wherein the Neisseria species bacterium produces alevel of a NMB0964 polypeptide sufficient to provide for production of avesicle that, when administered to a subject, elicits anti-NMB0964antibodies; and a pharmaceutically acceptable excipient.

This may be achieved due to the Neisseria species bacterium beinggenetically modified in NMB0964 polypeptide production by for instance:disrupting the functional expression of the Zur repressor (NMB1266)—aprotein which switches off expression of NMB0964 in the presence of Zn²⁺in the medium; replacing the NMB0964 promoter with one that does notbind Zur, in particular with a stronger promoter than the endogenousNMB0964 promoter such as a lac promoter; or through using a medium lowin Zn²⁺ concentration—i.e. under 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 μM free Zn²⁺—(such as Roswell ParkMemorial Institute medium 1640 (RPMI) which has around 1.69 μM Zn²⁺ byICP-MS), or removing Zn²⁺ in the medium, for instance using a known zincchelator such as TPEN(N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine)—enough should beadded to the medium such that the expression of the NMB0964 ismaximised.

The Neisseria species bacterium may be deficient in capsularpolysaccharide, for instance through disruption of functional expressionof the siaD gene. It may be disrupted in the functional expression ofthe msbB and/or htrB genes to detoxify the LOS in the outer membranevesicle. It may be disrupted in the expression of one or more thefollowing genes: PorA, PorB, OpA, OpC, PilC, or FrpB. It may bedisrupted in the functional expression of the IgtB gene. Such disruptionmethods are described in WO 01/09350 and WO2004/014417. The Neisseriaspecies bacterium may be of immunotype L2 or L3.

Methods for the preparation or isolation of outer membrane vesicles(also known as microvesicles or blebs) from Neisserial strains are wellknown in the art, and are described in WO 01/09350 and WO2004/014417.Typically outer membrane vesicles are isolated by extracting eitherwithout a detergent, or with 0-0.5, 0.02-0.4, 0.04-0.3, 0.06-0.2, or0.08-0.15 detergent, for instance deoxycholate, e.g. with around orexactly 0.1% deoxycholate.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Detection of Tdfl on Western blot. (FIG. 1A) HB-1 grown in TSB(lane 1), RPMI (lane 2) and the tdfl knockout strain grown in RPMI (lane3). (FIG. 1B) HB-1 grown in RPMI with increasing amounts of TSB added.(FIG. 1C) HB-1 grown in RPMI (lane 1), supplemented with 0.5 μM zinc(lane 2) or 1 μM zinc (lane 4). (FIG. 1D) HB-1 grown in RPMI (lane 1),with increasing concentrations of TPEN (0.1, 0.5 and 1 μM in lanes 2-4,respectively)

FIG. 2 (FIG. 2). Tdfl expression in wild type and zur mutant strains.The presence of Tdfl in cell lysates of HB-1 and the zur mutant grown inRPMI, RPMI with 600 nM zinc or TSB was assessed by Western blotanalysis.

FIG. 3. Topology model of Tdfl. FIG. 3A. The plug domain is colored darkgrey, the beta strands light gray and the extracellular loops white. Thehistidine/aspartic acid stretches are boxed. FIG. 3B. Ribbon structureof Tdfl.

FIG. 4. Zinc binding and transport by Tdfl. (FIG. 4.A) Zinc binding toouter membrane vesicles either containing or not Tdfl was measured by aPAR competition assay (FIG. 4B) Intracellular zinc concentrations asmeasured by ICP-MS of the wild-type strain, the tdfl mutant and the tonemutant. FIG. 4C. Zinc regulation of Tdfl is highly conserved inmeningococci. Western blot of cell lysates of the indicated strainsgrown in RPMI with or without added zinc. ^(a) Clonal group designationstaken from (36);—indicates that the strain was typed by Multi-LocusEnzyme Electrophoresis but could not be assigned to a specific clone.

FIG. 5. Protein profile of the Tdfl vaccine. Outer membrane vesiclesused to immunize mice for antiserum production were separated bySDS-PAGE and stained with Coomassie brilliant blue.

FIG. 6. Tdfl and PorB.

FIG. 7 (FIG. 7). Impact of IPTG on expression of Tdfl on cells used inSBA. See Example 1.

FIG. 8 (FIGS. 8A-B). Amino acid sequence alignment of Tdfl of N.meningitidis strains MC58 with those of 053422, FAM18 and Z2491, thecarrier strains α14, α153 and α275 The TonB box (Tb), the plug domain,the loops and the transmembrane domains (Tm) are marked above thesequence and the His- and Asp-rich stretches are underlined.

FIG. 9 (FIGS. 9A-C). Amino acid sequence alignment of the Tdflhomologues. The histidine aspartic acid rich stretches are highlightedin grey.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that an OMV vaccineprepared either in specific culture conditions low in Zn2+, or from amutant N. meningitidis strain engineered to either over-express NMB0964or to remove the Zinc repression mechanism mediated through Zur, isenriched in NMB0964, and such OMVs may elicit good bactericidal antibodyresponses compared to OMVs which have not been prepared with thesemethods.

By the term NMB0964 polypeptide herein it includes the neisserial Tdflpolypeptide (encoded by the tdfl gene) in general from any neisserialstrain (the protein is so well conserved amongst neisserial strains itsidentity in any particular neisserial strain is readily ascertainable bypersons skilled in the art). The term therefore includes the NMA1161sequence, and the BASB082 polypeptide sequence (and all the Polypeptidesof the Invention concerning the BASB082 polypeptide) of WO 00/55327. Forinstance the NMB0964 polypeptide of the invention will cover SEQ ID NO:2 of WO00/55327 or polypeptides with more than 70, 80, 90 or 95%sequence identity with said SEQ ID NO:2, or polypeptides comprisingimmunogenic fragments of 7, 10, 12, 15 or 20 (or more) contiguous aminoacids from said SEQ ID NO: 2 (in particular said immunogenic fragmentsbeing capable of eliciting—if necessary when coupled to a proteincarrier—an immune response which can recognise said SEQ ID NO: 2).Particularly preferred NMB0964 immunogenic fragment embodiments arethose extracellular loop sequences shown in the topology diagram of FIG.3 as applied to any given NMB0964 sequence. In particular the thirdextracellular loop is provided (wherein the 2 Cys residues areoptionally disulphide linked or not). Said NMB0964 immunogenic fragmentpolypeptide sequences may have more than 70, 80, 90 or 95% sequenceidentity with said extracellular loop sequences (as defined in FIG. 3)from SEQ ID NO:2 of WO 00/55327, or may be polypeptides comprisingimmunogenic fragments of 7, 10, 12, 15 or 20 (or more) contiguous aminoacids from said extracellular loop sequences (as defined in FIG. 3) fromSEQ ID NO: 2 (in particular said immunogenic fragments being capable ofeliciting—if necessary when coupled to a protein carrier—an immuneresponse which can recognise said SEQ ID NO: 2) and are provided asNMB0964 polypeptides of the invention. Said NMB0964 immunogenic fragmentpolypeptide sequences may have more than 70, 80, 90, 95, 99 or 100%sequence identity with the sequence from the third extracellular loopsequence given in FIG. 3 (wherein optionally the 2 Cys residues shouldbe conserved, and may or may not be disulphide linked), or may bepolypeptides comprising immunogenic fragments of 7, 10, 12, 15 or 20 (ormore) contiguous amino acids from said extracellular loop sequence (inparticular said immunogenic fragments being capable of eliciting—ifnecessary when coupled to a protein carrier—an immune response which canrecognise SEQ ID NO: 2 of WO00/55327) and are provided as NMB0964polypeptides of the invention. In one embodiment the NMB0964 immunogenicfragment polypeptides are not full-length NMB0964 (mature sequence orwith signal sequence) polypeptides. Thus a further aspect of theinvention is a immunogenic composition comprising such NMB0964immunogenic fragment polypeptide sequences of the invention and apharmaceutically acceptable excipient.

The term “a level of a NMB0964 polypeptide sufficient to provide forproduction of a vesicle that, when administered to a subject, elicitsanti-NMB0964 antibodies” in one embodiment indicates that the level issufficient to induce detectable bactericidal antibodies, for instanceSBA titres of 100 or more, for instance it indicates that 5 μg totalprotein content outer membrane vesicles of the invention whenintramuscularly injected into mice at days 0, 21 and 28 produces serumon day 42 which generates an SBA titre of over 100 (for instance greaterthan 150, 200, 250, 300, 350, 400, 500, 700, 900 or 1000) using the SBAassay in the “Serum Bactericidal Assay” section of Example 2.

The heterologous promoter associated with the polypeptide of theinvention being “stronger” than the non-repressed endogenous promoter ofthe polypeptide of the invention means that its use results in theexpression of more polypeptide of the invention than when anon-repressed endogenous promoter of the polypeptide of the invention isutilised.

The term “protective immunity” means that a vaccine or immunizationschedule that is administered to a mammal induces an immune responsethat prevents, retards the development of, or reduces the severity of adisease that is caused by Neisseria meningitidis, or diminishes oraltogether eliminates the symptoms of the disease.

The phrase “a disease caused by a strain of serogroup B of Neisseriameningitidis” encompasses any clinical symptom or combination ofclinical symptoms that are present in an infection with a member ofserogroup B of Neisseria meningitidis. These symptoms include but arenot limited to: colonization of the upper respiratory tract (e.g. mucosaof the nasopharynx and tonsils) by a pathogenic strain of serogroup B ofNeisseria meningitidis, penetration of the bacteria into the mucosa andthe submucosal vascular bed, septicemia, septic shock, inflammation,haemorrhagic skin lesions, activation of fibrinolysis and of bloodcoagulation, organ dysfunction such as kidney, lung, and cardiacfailure, adrenal hemorrhaging and muscular infarction, capillaryleakage, edema, peripheral limb ischaemia, respiratory distresssyndrome, pericarditis and meningitis.

“Serogroup” as used herein refers to classification of Neisseriameningitides by virtue of immunologically detectable variations in thecapsular polysaccharide. About 12 serogroups are known: A, B, C, X, Y,Z, 29-E, W-135, H, I, K and L. Any one serogroup can encompass multipleserotypes and multiple serosubtypes.

“Enriched” means that an antigen in an antigen composition ismanipulated by an experimentalist or a clinician so that it is presentin at least a three-fold greater concentration by total weight, usuallyat least 5-fold greater concentration, more preferably at least 10-foldgreater concentration, or at least 100-fold greater concentration thanthe concentration of that antigen in the strain from which the antigencomposition was obtained. Thus, if the concentration of a particularantigen is 1 microgram per gram of total bacterial preparation (or oftotal bacterial protein), an enriched preparation would contain at least3 micrograms per gram of total bacterial preparation (or of totalbacterial protein).

The NMB0964 polypeptide of the invention may be enriched in the outermembrane vesicles of the invention through the methods discussed herein(for instance the culture conditions, or the overexpression of thepolypeptide through recombinant means).

The term “heterologous” refers to two biological components that are notfound together in nature. The components may be host cells, genes, orregulatory regions, such as promoters. Although the heterologouscomponents are not found together in nature, they can function together,as when a promoter heterologous to a gene is operably linked to thegene. Another example is where a Neisserial sequence is heterologous toa Neisserial host of a different strain. “Heterologous” as used hereinin the context of proteins expressed in two different bacterial strains,indicates that the proteins in question differ in amino acid sequence.

The production strain can be a capsule deficient strain. Capsuledeficient strains can provide vesicle-based vaccines that provide for areduced risk of eliciting a significant autoantibody response in asubject to whom the vaccine is administered (e.g., due to production ofantibodies that cross-react with sialic acid on host cell surfaces).“Capsule deficient” or “deficient in capsular polysaccharide” as usedherein refers to a level of capsular polysaccharide on the bacterialsurface that is lower than that of a naturally-occurring strain or,where the strain is genetically modified, is lower than that of aparental strain from which the capsule deficient strain is derived. Acapsule deficient strain includes strains that are decreased in surfacecapsular polysaccharide production by at least 10%, 20%, 25%, 30%, 40%,50%, 60%, 75%, 80%, 85%, 90% or more, and includes strains in whichcapsular polysaccharide is not detectable on the bacterial surface(e.g., by whole cell ELISA using an anti-capsular polysaccharideantibody).

Capsule deficient strains include those that are capsule deficient dueto a naturally-occurring or recombinantly-generated geneticmodification. Naturally-occurring capsule deficient strains (see, e.g.,Dolan-Livengood et al. J. Infect. Dis. (2003) 187(10): 1616-28), as wellas methods of identifying and/or generating capsule-deficient strains(see, e.g., Fisseha et al. (2005) Infect. Immun. 73(7):4070-4080;Stephens et al. (1991) Infect Immun 59(11):4097-102; Frosch et al.(1990) Mol Microbiol. 1990 4(7):1215-1218) are known in the art.

Modification of a Neisserial host cell to provide for decreasedproduction of capsular polysaccharide may include modification of one ormore genes involved in capsule synthesis, where the modificationprovides for, for example, decreased levels of capsular polysacchariderelative to a parent cell prior to modification. Such geneticmodifications can include changes in nucleotide and/or amino acidsequences in one or more capsule biosynthesis genes rendering the straincapsule deficient (e.g., due to one or more insertions, deletions,substitutions, and the like in one or more capsule biosynthesis genes).Capsule deficient strains can lack or be non-functional for one or morecapsule genes. Of particular interest are strains that are deficient insialic acid biosynthesis.

Such strains can provide for production of vesicles that have reducedrisk of eliciting anti-sialic acid antibodies that cross-react withhuman sialic acid antigens, and can further provide for improvedmanufacturing safety. Strains having a defect in sialic acidbiosynthesis (due to either a naturally occurring modification or anengineered modification) can be defective in any of a number ofdifferent genes in the sialic acid biosynthetic pathway. Of particularinterest are strains that are defective in a gene product encoded by theN-acetylglucosamine-6-phosphate 2-epimerase gene (known as synXAAF40537.1 or siaA AAA20475), with strains having this gene inactivatedbeing of especial interest. For example, in one embodiment, a capsuledeficient strain is generated by disrupting production of a functionalsynX gene product (see, e.g., Swartley et al. (1994) J Bacteriol.176(5):1530-4).

Capsular deficient strains can also be generated fromnaturally-occurring strains using non-recombinant techniques, e.g., byuse of bactericidal anti-capsular antibodies to select for strains thatreduced in capsular polysaccharide.

In general as noted above, vesicles can be produced according to theinvention using a naturally-occurring or modifiednon-naturally-occurring Neisserial strain that produces vesicles withsufficient NMB0964 protein that, when administered to a subject, providefor production of anti-NMB0964 antibodies.

In one embodiment, the Neisserial strain used to produce vesiclesaccording to the invention can be naturally occurring strains thatexpress a higher level of NMB0964 relative to strains that express nodetectable or a low level of NMB0964.

In another embodiment, the Neisserial strain is modified by recombinantor non-recombinant techniques to provide for a sufficiently high levelof NMB0964 production.

Such modified strains generally are produced so as to provide for anincrease in NMB0964 production that is 1.5, 2, 2.5 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or greater over NMB0964production in the unmodified parental cell or over NMB0964 production ofthe strain RM1O9O or H44/76. Any suitable strain can be used in thisembodiment, including strains that produce low or undetectable levels ofNMB0964 prior to modification and strains that naturally produce highlevels of NMB0964 relative to strains that express no detectable or alow level of NMB0964.

Modified strains may be produced using recombinant techniques, usuallyby introduction of nucleic acid encoding a NMB0964 polypeptide ormanipulation of an endogenous NMB0964 gene to provide for increasedexpression of endogenous NMB0964.

As noted above, this may be done by introduction of nucleic acidencoding a NMB0964 polypeptide or manipulation of an endogenous NMB0964gene to provide for increased expression of endogenous NMB0964.

Endogenous NMB0964 expression can be increased by altering in situ theregulatory region controlling the expression of NMB0964. Methods forproviding for increased expression of an endogenous Neisserial gene areknown in the art (see, e.g., WO 02/09746).

Modification of a Neisserial host cell to provide for increasedproduction of endogenous NMB0964 may include partial or totalreplacement of all of a portion of the endogenous gene controllingNMB0964 expression, where the modification provides for, for example,enhanced transcriptional activity relative to the unmodified parentalstrain.

Increased transcriptional activity may be conferred by variants (pointmutations, deletions and/or insertions) of the endogenous controlregions, by naturally occurring or modified heterologous promoters or bya combination of both. In general the genetic modification confers atranscriptional activity greater than that of the unmodified endogenoustranscriptional activity (e.g., by introduction of a strong promoter),resulting in an enhanced expression of NMB0964.

Typical strong promoters that may be useful in increasing NMB0964transcription production can include, for example, the promoters ofporA, porB, lbpB, tbpB, p110, hpuAB, lgtF, Opa, p110, lst, and hpuAB.PorA, RmpM and PorB are of particular interest as constitutive, strongpromoters. PorB promoter activity is contained in a fragmentcorresponding to nucleotides −1 to −250 upstream of the initiation codonof porB.

Methods are available in the art to accomplish introduction of apromoter into a host cell genome so as to operably link the promoter toan endogenous NMB0964-encoding nucleic acid. For example, doublecross-over homologous recombination technology to introduce a promoterin a region upstream of the coding sequence, e.g., about 1000 bp, fromabout 30-970 bp, about 200-600 bp, about 300-500 bp, or about 400 bpupstream (5′) of the initiation ATG codon of the NMB0964-encodingnucleic acid sequence to provide for up-regulation. Optimal placement ofthe promoter can be determined through routine use of methods availablein the art.

For example, a highly active promoter (e.g., PorA, PorB or RmpMpromoters) upstream of the targeted gene. As an example, the PorApromoter can be optimized for expression as described by van der Ende etal. Infect Immun 2000; 68:6685-90. Insertion of the promoter can beaccomplished by, for example, PCR amplification of the upstream segmentof the targeted NMB0964 gene, cloning the upstream segment in a vector,and either inserting appropriate restriction sites during PCRamplification, or using naturally occurring restriction sites to insertthe PorA promoter segment. For example, an about 700 bp upstream segmentof the NMB0964 gene can be cloned. Using naturally occurring restrictionenzyme sites located at an appropriate distance (e.g., about 400 bp)upstream of the NMB0964 promoter within this cloned segment a PorApromoter segment is inserted. An antibiotic (e.g., erythromycin)resistance cassette can be inserted within the segment further upstreamof the PorA promoter and the construct may be used to replace thewild-type upstream NMB0964 segment by homologous recombination.

Another approach involves introducing a NMB0964 polypeptide-encodingsequence downstream of an endogenous promoter that exhibits strongtranscriptional activity in the host cell genome. For example, thecoding region of the RmpM gene can be replaced with a coding sequencefor a NMB0964 polypeptide. This approach takes advantage of the highlyactive constitutive RmpM promoter to drive expression.

Neisserial strains can be genetically modified to over-express NMB0964by introduction of a construct encoding a NMB0964 polypeptide into aNeisserial host cell. The NMB0964 introduced for expression is referredto herein as an “exogenous” NMB0964. The host cell produces anendogenous NMB0964, the exogenous NMB0964 may have the same or differentamino acid sequence compared to the endogenous NMB0964.

The NMB0964 polypeptides useful in the invention also include fusionproteins, where the fusion protein comprises a NMB0964 polypeptidehaving a fusion partner at its N-terminus or C-terminus. Fusion partnersof interest include, for example, glutathione S transferase (GST),maltose binding protein (MBP), His-tag, and the like, as well as leaderpeptides from other proteins.

Sequence identity can be determined using methods for alignment andcomparison of nucleic acid or amino acid sequences, which methods arewell known in the art. Comparison of longer sequences may require moresophisticated methods to achieve optimal alignment of two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needlemanand Wunsch (1970) J Mol. Biol. 48:443, by the search for similaritymethod of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA)85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e. resulting in the highestpercentage of sequence similarity over the comparison window) generatedby the various methods is selected.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appi. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BBSTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (www.ncbi.nlm.nih.govl). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra).

These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences), uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Nati. Acad. Sci. USA 89:10915(1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptidesshare sequence identity is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below.

Thus, a polypeptide typically share sequence identity with a secondpolypeptide, for example, where the two polypeptides differ only byconservative substitutions. Another indication that two nucleic acidsequences share sequence identity is that the two molecules hybridize toeach other under stringent conditions. The selection of a particular setof hybridization conditions is selected following standard methods inthe art (see, for example, Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Anexample of stringent hybridization conditions is hybridization at 50° C.or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate).Another example of stringent hybridization conditions is overnightincubation at 42° C. in a solution: % formamide, 5×SSC (150 mM NaC1, 15nlM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

Stringent hybridization conditions are hybridization conditions that areat least as stringent as the above representative conditions, whereconditions are considered to be at least as stringent if they are atleast about 80% as stringent, typically at least about 90% as stringentas the above specific stringent conditions. Other stringenthybridization conditions are known in the art and may also be employedto identify nucleic acids of this particular embodiment of theinvention.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Methods and compositions which can be readily adapted to provide forgenetic modification of a Neisserial host cell to express an exogenousNMB0964 polypeptide are known in the art. Exemplary vectors and methodsare provided in WO 02/09746 and O'Dwyer et al. Infect Immun 2004; 72:6511-80.

Methods for transfer of genetic material into a Neisserial host include,for example, conjugation, transformation, electroporation, calciumphosphate methods and the like. The method for transfer should providefor stable expression of the introduced NMB0964 encoding nucleic acid.The NMB0964-encoding nucleic acid can be provided as a inheritableepisomal element (e.g., plasmid) or can be genomically integrated.

Suitable vectors will vary in composition depending what type ofrecombination event is to be performed. Integrative vectors can beconditionally replicative or suicide plasmids, bacteriophages,transposons or linear DNA fragments obtained by restriction hydrolysisor PCR amplification. Selection of the recombination event can beaccomplished by means of selectable genetic marker such as genesconferring resistance to antibiotics (for instance kanamycin,erythromycin, chloramphenicol, or gentamycin), genes conferringresistance to heavy metals and/or toxic compounds or genes complementingauxotrophic mutations (for instance pur, leu, met, aro).

In one embodiment, the vector is an expression vector based on episornalplasmids containing selectable drug resistance markers that autonomouslyreplicate in both E. coli and N. meningitidis. One example of such a“shuttle vector” is the plasmid pFP1O (Pagotto et al. Gene 2000244:13-19).

Immunization

In general, the methods of the invention provide for administration ofone or more antigenic compositions of the invention to a mammaliansubject (e.g., a human) so as to elicit a protective immune responseagainst more than one strain of Neisseria species bacteria, and thusprotection against disease caused by such bacteria. In particular, themethods of the invention can provide for an immunoprotective immuneresponse against a 1, 2, 3, 4, or more strains of Neisseria meningitidisspecies, where the strains differ in at least one of serogroup,serotype, serosubtype, or NMB0964 polypeptide. Of particular interest isinduction of a protective immune response against multiple strains ofNeisseria meningitidis of serogroup B, particularly where the strainsdiffer in serosubtype (e.g., have heterologous PorAs). Also ofparticular interest is induction of a protective immune response againststrains that are heterologous to one other in terms of PorA and/orNMB0964.

The antigenic compositions of the invention can be administered orally,nasally, nasopharyngeally, parenterally, enterically, gastrically,topically, transdermally, subcutaneously, intramuscularly, in tablet,solid, powdered, liquid, aerosol form, locally or systemically, with orwithout added excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemingtons Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980).

It is recognized that oral administration can require protection of thecompositions from digestion. This is typically accomplished either byassociation of the composition with an agent that renders it resistantto acidic and enzymatic hydrolysis or by packaging the composition in anappropriately resistant carrier. Means of protecting from digestion arewell known in the art.

The compositions are administered to an animal that is at risk fromacquiring a Neisserial disease to prevent or at least partially arrestthe development of disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for therapeutic use will depend on, e.g., theantigenic composition, the manner of administration, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician. Single or multiple doses of the antigeniccompositions may be administered depending on the dosage and frequencyrequired and tolerated by the patient, and route of administration.

The antigenic compositions (herein also known as immunogeniccompositions) described herein can comprise a mixture of vesicles whichvesicles can be from the same or different strains. In anotherembodiment, the antigenic compositions can comprise a mixture ofvesicles from 2, 3, 4, 5 or more strains.

The antigenic compositions are administered in an amount effective toelicit an immune response, particularly a humoral immune response, inthe host. Amounts for the immunization of the mixture generally rangefrom about 0.001 mg to about 1.0 mg per 70 kilogram patient, morecommonly from about 0.001 mg to about 0.2 mg per 70 kilogram patient.Dosages from 0.001 up to about 10 mg per patient per day may be used,particularly when the antigen is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages (e.g. 10 to 100 mg or more) arepossible in oral, nasal, or topical administration. The initialadministration of the mixture can be followed by booster immunization ofthe same of different mixture, with at least one booster, more usuallytwo boosters, being preferred.

The antigen compositions are typically administered to a mammal that isimmunologically naïve with respect to Neisseria, particularly withrespect to Neisseria meningitidis. In a particular embodiment, themammal is a human child about five years or younger, and preferablyabout two years old or younger, and the antigen compositions areadministered at any one or more of the following times: two weeks, onemonth, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or one year or 15, 18,or 21 months after birth, or at 2, 3, 4, or 5 years of age.

In general, administration to any mammal is preferably initiated priorto the first sign of disease symptoms, or at the first sign of possibleor actual exposure to Neisseria.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Example 1 Immunogenicity of OMVs with Up-Regulation of Tdfl

Tdfl is a gene which is thought to be expressed when N. meningitidis iswithin the blood. It is therefore not normally expressed when strainsare grown in conventional culture media, but wild-type strain H44/76,for example, can be made to express the protein in special cultureconditions (RPMI culture media supplemented with hemin). The followingexperiment details the use of an H44/76 strain where Tdfl expression hasbeen recombinantly made inducible (through the use of IPTG). This allowsthe over-expression of Tdfl on the surface of OMV vaccines made from thestrain, and provides an easy way of culturing a strain expressing theantigen to establish whether antibodies generated against Tdfl arecapable of killing such a modified strain which expresses Tdfl undernormal culture conditions (+IPTG). The impact of IPTG on expression ofTdfl on cells used in the SBA is shown in FIG. 7.

Groups of 10 mice were immunized three times with OMV by theintramuscular route on day 0, 21 and 28. Each inoculation was made up of5 μg (protein content) of OMVs formulated on AIPO4 with MPL. The OMVswere derived from Neisseria meningitidis strain H44/76, engineered sothat capsular polysaccharides and PorA were down regulated and LOSimmunotype was galE type. A comparison was made of OMVs in which Tdflwas or was not up-regulated (up-regulation under the control of IPTGinducible promoter). On day 42, blood samples were taken for analysis byserum bactericidal assay using either the homologous strain H44/76(B:15:P1.7,16) expressing or not Tdfl (after addition or not of IPTG inthe culture media).

N. meningitidis strains were cultivated overnight on GC-agar with 10μg/ml chloramphenicol Petri Dishes at 37° C.+5% CO₂. They weresub-cultured for 3 hours in a liquid TSB medium supplemented or not withIPTG 1000 μM. Individual sera were inactivated for 30 min at 56° C.Serum samples were diluted in HBSS-BSA 0.5% and then twofold diluted (8dilutions) in a volume of 25 μl in flat bottom microplates. Bacteriawere diluted in HBSS-BSA 0.5% to yield 8·10³ CFU/ml and 12.5 μl of thisdilution was added to the serum dilution. Rabbit complement (12.5 μl)was also added to each well. After 75 min of incubation at 37° C. undershaking, 15 ul of the mixture was spread onto pre-warmed GC-agar platesincubated overnight at 37° C.+CO₂.

The CFU's were counted and the percentage of killing was calculated. TheSBA titer is the dilution giving 50% of killing.

SBA Titers: Impact of Expression of Tdfl by Target Cells

SBA titers H44/76 without IPTG <50 H44/76 with IPTG 400; 400; 800

Without IPTG, Tdfl is not expressed on target cells which are not killedby sera from mice immunized with up-regulated Tdfl OMVs. When theexpression of Tdfl is specifically induced by IPTG the target cellsexpress Tdfl and are killed by anti-Tdfl-OMVs mice sera.

Example 2 A Novel Zinc-Regulated Outer Membrane Protein in Neisseriameningitidis with Vaccine Potential Abstract

Since the concentration of free iron in the human host is low, efficientiron-acquisition mechanisms constitute important virulence factors forpathogenic bacteria. In the Gram-negative bacteria, TonB-dependent outermembrane receptors are implicated in iron acquisition. However,transport across the bacterial outer membrane of other metals that arealso scarce in the human host is far less clear. In this study wecharacterized a novel TonB-dependent receptor in Neisseria meningitidis.We show that the bacteria produce this protein under zinc limitation andthat it is involved in zinc uptake. Furthermore, since the protein ishighly conserved among isolates and is capable of inducing bactericidalantibodies, it constitutes a novel candidate for the development of avaccine against N. meningitidis for which no effective universal vaccineis available so far. Homologues of the protein, designated Tfdl, arefound in many other pathogens residing in the respiratory tract,suggesting that receptor-mediated zinc uptake is particularly importantfor survival in this niche.

Introduction

The cell envelope of Gram-negative bacteria consists of two membranes,the inner and the outer membrane, which are separated by the periplasmcontaining the peptidoglycan layer. The outer membrane forms a barrierfor harmful compounds from the environment. Most nutrients can pass theouter membrane by passive diffusion via abundant channel-forming outermembrane proteins, collectively called porins. However, diffusion is notan option when the extracellular concentration of a nutrient is low.This is the case, for example, for iron. Pathogens are confronted withlow concentrations of free iron within the human host, where iron isbound by iron-transport and—storage proteins, such as lactoferrin andtransferrin. Hence, efficient iron acquisition mechanisms constituteimportant virulence factors and have been studied extensively in manypathogens (1, 2).

When grown under iron-limiting conditions, Gram-negative bacteria inducethe synthesis of outer membrane proteins that function as receptors forthe iron-binding proteins of the host, for heme, or for siderophores,which are small iron-chelating compounds produced and secreted by thebacteria under iron limitation. The resolved crystal structures of suchreceptors revealed 22-stranded β-barrels, which do not form openchannels but are closed by an N-terminal plug domain (3). After bindingof the ligand to the receptor, the subsequent uptake is an activeprocess that requires the energy of the proton gradient across the innermembrane, which is coupled to the receptors in the outer membrane via acomplex of three proteins, the TonB complex (4, 5).

While iron-acquisition mechanisms have been studied extensively in manyGram-negative bacteria, little is known yet about the transport of otheressential heavy metals, such as zinc and manganese, across the bacterialouter membrane. The concentration of these trace elements also is low inthe human host, which, for example, responds to infections by theproduction of metallothioneins and calprotectin thereby reducing theavailability of metals to the invading pathogens (6, 7). Therefore,Gram-negative pathogens likely possess effective acquisition mechanismsfor these metals, which may or may not resemble the iron-acquisitionsystems.

Neisseria meningitidis is an obligate human pathogen that can colonizethe nasopharyngeal mucosa asymptomatically. Occasionally the bacteriumenters the bloodstream and can cause meningitis and sepsis with a highmortality rate (8). While vaccines are available for most pathogenicserogroups of N. meningitidis based on the capsular polysaccharides, avaccine against serogroup B meningococci is lacking. The polysaccharidecapsule of the serogroup B strains is poorly immunogenic due to itsresemblance to human glycoproteins (9). Thus, subcapsular antigens arebeing studied as alternative vaccine components; however, these studiesare frustrated by the high antigenic variability of the major outermembrane proteins. Therefore, attention has shifted to minor antigens,including the TonB-dependent receptors.

When grown under iron limitation, N. meningitidis producesTonB-dependent receptors for lactoferrin (10), transferrin (11),hemoglobin (12, 13) and enterobactin (14), all involved in the uptake ofiron. Based on homology searches, Turner et al (15) identified sevenadditional genes for putative TonB-dependent family (Tdf) members in theavailable genome sequences of three Neisserial strains. Interestingly,the expression of some of these tdf genes appeared unaffected by ironavailability in various microarray studies (16, 17), indicating thattheir products might be implicated in the transport of metals other thaniron. Here we studied the regulation of the synthesis, the function andthe vaccine potential of one of these receptors and show that thisreceptor is involved in the uptake of zinc.

Results Tdfl is not a Heme Receptor

Tdfl (locus tags NMA1161 and NMB0964 in the sequenced genomes of N.meningitidis serogroup A strain Z2491 and serogroup B strain MC58,respectively) was previously identified as one of seven novel putativeTonB-dependent receptors present in the Neisserial genomes (15) and wasfound to be up-regulated in the presence of naïve human serum (18).Since almost all TonB-dependent receptors studied to date are involvedin iron acquisition we assumed that Tdfl transports an iron complex.This idea was strengthened by the fact that blast searches (19) with theamino-acid sequence of NMA1161 revealed high sequence similarity toouter membrane receptors for the uptake of heme, such as HumA ofMoraxella catarrhalis (20) with 41% identity and 58% similarity.

To assess the function of Tdfl, we constructed a tdfl deletion mutant ofa non-encapsulated derivative of serogroup B strain H44/76 called HB-1.We found similar binding of heme to HB-1 and the tdfl mutant as assessedby dot blot analysis and the tdfl mutant strain could still grow onplates with heme as the sole iron source. We could also not findincreased heme binding by Escherichia coli cells expressing Tdfl. Alsowe were unable to complement an E. coli heme auxotroph (data not shown).Therefore, we hypothesized that Tdfl, although homologous to hemereceptors, does not function as a heme receptor.

Regulation of tdfl by Zinc

Since Tdfl is not a heme receptor and is not found to be regulated byiron, we sought conditions where we could detect tdfl is expression inthe capsule deficient H44/76 Neisseia meningitidis HB-1. We could neverdetect Tdfl on Western blots when the bacteria were grown in tryptic soybroth (TSB), a complex rich medium (FIG. 1 A, lane 1). However, when thebacteria were grown in the chemically defined RPMI medium, Tdfl wasdetectable in bacterial lysates (FIG. 1 A, lane 2). The specificity ofthe signal detected was demonstrated by its absence in the tdfl knockoutstrain grown in RPMI (FIG. 1 A, lane 3). We noted that the presence ofeven small amounts of TSB added to RPMI negatively affected Tdflsynthesis (FIG. 1 B); apparently TSB contains a compound that repressesthe transcription of tdfl. Since we noticed that RPMI does not contain asource of trace metals, we decided to test whether addition of acocktail of trace metals, containing cobalt, molybdenum, manganese,copper and zinc, would repress tdfl expression, which indeed appeared tobe the case. We then tested all these metals separately and found thatspecifically zinc, even at sub-μM concentrations, caused repression oftdfl expression (FIG. 1 C). Since standard RPMI is not supplemented witha specific zinc source, the available zinc required for bacterial growthpresumably comes from the water and/or traces in the salts used to makethe medium. We measured the zinc concentration in RPMI medium byinductively coupled plasma mass spectrometry (ICP-MS) and found it to be˜110 parts per billion (˜1.69 μM).

The zinc regulation of tdfl became even more evident when wesupplemented the RPMI medium with the specific zinc chelatorN,N,N′,N′-Tetrakis-(2-pyridylmethyl)-Ethylenediamine (TPEN). Addition ofTPEN to the medium resulted in a dose-dependent increase in Tdflsynthesis (FIG. 1 D). However, concentrations above 1 μM TPEN totallyinhibited cell growth presumably due to total zinc depletion from themedium. Growth could be restored by the addition of zinc (data notshown). The zinc regulation of tdfl was confirmed by real-timequantitative PCR (RT-qPCR) using total RNA obtained from cultures grownin RPMI supplemented or not with 500 nM zinc or 0.5 μM TPEN. The datashowed a 13.8-fold repression in the presence of zinc and a 3.8-fold upregulation in the presence of TPEN. The fold difference between addedTPEN and zinc was 52.6-fold.

Role of the Transcriptional Regulator Zur in tdfl Expression

In E. coli, the zinc uptake regulator (Zur) has been shown to regulatethe expression of the znuACB genes, which encode the periplasmic bindingprotein, the ATPase and the integral inner membrane component requiredfor zinc transport from the periplasm to the cytoplasm (23). In thepresence of zinc, Zur binds a Zur-binding element (consensusGAAATGTTATANTATAACATTTC) (SEQ NO:1) in the promoter of the znuACB operonand thereby blocks transcription.

In the genome sequence of N. meningitidis strain MC58, we identifiedhomologues of the E. coli zur gene, i.e. NMB1266, and of znuCBA, i.e.NMB0588, NMB0587, and NMB0586. In addition, we found sequencesresembling the E. coli Zur binding consensus in the regions upstream ofthe neisserial tdfl (GtAATGTTATATaATAACAaact) (SEQ NO:2) and znuC(cAAAcGTTATACagTAtCATaTC) (SEQ NO:3) (identical nucleotides to the E.coli consensus are in capital case). To confirm the involvement of Zurin the regulation of tdfl expression, we generated a zur mutant ofstrain HB-1, which, indeed, produced Tdfl constitutively (FIG. 2). Also,RT-qPCR demonstrated the involvement of Zur in the expression of znuAand tdfl as znuA and tdfl expression levels increased 5- and 34-fold,respectively, in the zur mutant compared to its parent strain both grownin the presence of zinc.

Tdfl Facilitates Zinc Acquisition

Since the expression of tdfl is regulated by the availability of zinc,it is likely that Tdfl acts as a receptor for zinc or a zinc-containingcomplex. We first analyzed the amino acid sequence and constructed atopology model of Tdfl using the PROFtmb program at www.rostlab.org,(FIG. 3). Tdfl contains two cysteine residues in the putativeextracellular loop L3. If these cysteines form a disulfide bond(supported by our analysis of the membrane fraction of bacteria bySDS-PAGE with and without DTT where incubation of the sample with thereducing agent resulted in a shift in electrophoretic mobility,presumably due to the disruption of the disulfide bond), they bring twostretches of amino acid residues, both rich in histidine and asparticacid residues, in close proximity (FIG. 3), which could be of functionalimportance, since also in the periplasmic ZnuA protein of E. coli, astretch of His and Asp residues is involved in binding zinc (25). Thus,we considered the possibility that Tdfl binds free zinc and transportsit to the periplasm. To test this hypothesis we first determined whetherTdfl could bind zinc. We compared outer membrane vesicles with andwithout Tdfl for their ability to compete with4-(2-pyridylazo)resorcinol (PAR) for zinc. The outer membrane vesiclescontaining Tdfl showed ˜40% increased binding of zinc compared to thevesicles without Tdfl (FIG. 4A). To test transport of zinc we comparedthe tdfl knockout, a tonB knockout and their parent strain for theaccumulation of intracellular zinc using ICP-MS. HB-1 accumulated ˜33%more zinc than the tdfl mutant or the tonB mutant, indicating that Tdfltransports free zinc and that this transport needs the TonB system (FIG.4B).

If indeed Tdfl is involved in the uptake of free zinc, than one wouldexpect derepression of znu gene expression to occur at higher externalzinc concentrations in the tdfl mutant as compared with the wild-typestrain. To test this idea, we grew the tdfl mutant and the parent strainin RPMI medium with 500 nM additional zinc, which largely, but notcompletely represses tdfl expression in the wild-type strain (FIG. 1 C).We subsequently measured the relative levels of tdfl and znuA mRNA byRT-qPCR. The tdfl mutant still contains the first 437 nucleotides of thetdfl gene that were used for the detection of gene expression. In thetdfl mutant, there was 18.6-fold more tdfl and 7.4-fold more znuAexpressed, showing that indeed the intracellular zinc concentration inthe tdfl mutant is lower than that in the parent strain under theapplied growth conditions. Also a znuA knockout strain expressed highlevels of Tdfl in the presence of zinc, confirming that ZnuA is requiredto sustain sufficient zinc levels in the cell (FIG. 4C). Thus, both Tdfland ZnuA are involved in the transport of zinc.

Conservation of Tdfl

Besides the function f Tdfl we also want to investigate whether Tdfl isa vaccine candidate for a universal N. meningitidis vaccine. One of thecriteria is that the antigen has to be conserved. We first looked at theavailable N. meningitidis genomes and found that Tdfl has a striking97-99% amino acid identity of the mature protein (FIG. 8). The sequencedifferences are scattered throughout the protein and are not clusteredin predicted extracellular loop regions, which are often antigenicallyvariable in Neisseria outer membrane proteins (FIG. 8). We subsequentlyanalyzed the presence of Tdfl in a panel of 32 different N. meningitidisisolates from different serogroups and different clonal lineages. Eachstrain was grown in RPMI medium supplemented or not with 500 nM zinc andanalyzed by Western blotting with the antiserum raised against Tdfl ofH44/76. All strains showed a repression of Tdfl in the presence of zinc(FIG. 5).

We then wanted to know the homology of Tdfl to other pathogenicbacteria. We first compared Tdfl with N. gonorrhea and found a 96%identity and a 97% similarity between these two Neisseria strains. Next,we used the blast program at NCBI with a cutoff of 40% identity at theamino acid level to search for homologs of Tdfl in other pathogenicbacteria. We identified homologs in other pathogenic bacteria, includingM. catarrhalis, Haemophilus parasuis, Mannheimia haemolytica,Acinetobacter baumannii, Pasteurella multocida, Bordetella pertussis andActinobacillus pleuropneumoniae, averaging a 41% identity and 59%similarity at the amino acid level and all Tdfl homologs have theHis/Asp region (FIG. 9). Interestingly, in B. pertussis the tdflhomologue is located adjacent to homologues of the znuABC and zur genes,again indicating a functional relationship between these genes.Furthermore, all these Tdfl homologs contain His- and Asp-rich stretches(FIG. 9).

Tdfl Induces Bactericidal Antibodies

To investigate the vaccine potential of Tdfl, we immunized mice withNeisserial outer membrane vesicles containing overexpression levels ofthis protein (FIG. 6A) and tested the resultant sera for the presence ofbactericidal antibodies. Routinely, we perform serum bactericidal assayson bacteria grown in TSB medium; however, under these conditions tdfl isnot expressed. Therefore, we tested the sera for bactericidal activityon a strain that expressed Tdfl from an isopropylβ-D-1-thiogalactopyranoside (IPTG)-inducible promoter and comparedcultures grown with and without IPTG. The bactericidal titers of thesera were <1:100 when IPTG was absent, but 1:1042 when IPTG was presentduring growth of the bacteria. Titers in pre-immune sera were also<1:100. These data clearly show that Tdfl is able to elicit bactericidalantibodies. We also wanted to investigate whether normalchromosome-encoded tdfl expression levels are sufficient to mediatecomplement-mediated killing. For this we employed the zur knockoutstrain that produces Tdfl constitutively in the TSB medium and growscomparable to the wild-type strain in this medium.

Discussion

The high-affinity ZnuABC uptake system for zinc has previously beenidentified in N. gonorrhoeae (30). Homologues can be found in themeningococcal genome, as described above, and in the genomes of manyother bacteria. In Salmonella enterica this ABC transporter has beenassociated with virulence (31). In no case, an outer membrane receptorinvolved in zinc acquisition has been identified and it is thought thatzinc diffuses through the porins.

In the human host, however, the free zinc levels are most likely too lowto sustain bacterial growth by passive diffusion. The total amount ofzinc in human serum is approximately 19 μM, but the vast majority isbound by serum proteins such as albumin (32). Here we have identified anouter membrane receptor, Tdfl that is regulated by zinc. The addition of700 nM zinc to the growth medium completely repressed Tdfl expression.The function of Tdfl is to bind and transport of unbound (free) zinc. Wepredict that the zinc is bound initially by the His/Asp stretch in theexternal loop and then internalized via two histidines that are on topof the plug domain (FIG. 3b ). A possible role for the TonB system inzinc uptake is that it pulls the plug out of the barrel and with thismovement the zinc bound to the two His residues is transported into theperiplasm where it is picked up by the periplasmic binding protein ZnuA.

Interestingly, similar regulation of tdfl and znuA expression wasreported in a microarray study using N. gonorrhoeae (33). The tdflhomolog NGO1205 and the znuA homolog NGO0168 were upregulated in amutant lacking the NGO0542 gene. This gene was annotated in that studyas perR because of its homology to a manganese-dependentperoxide-responsive regulator found in gram-positive organisms (34).However, this is the same gene we have annotated as zur. The zurannotation is clearly more accurate, because we show an identicalregulation by the absence of zur or the absence of zinc. More evidencefor the annotation zur rather than perR comes from the same study in N.gonorrhoeae. Microarrays performed with the gonococcal perR mutantshowed upregulation also of the ribosomal proteins L31 and L36. TheNeisserial genomes contain two copies for each of the genes encodingthese proteins with one form of each protein containing a zinc ribbonmotif. Zinc availability was found to be the key factor controlling thetype of L31/L36 protein expressed in B subtilis (34). In the gonococcalperR mutant, expression specifically of L31 and L36 paralogs lacking thezinc ribbons is induced, highly indicative of a disturbed zincregulation in a perR mutant. Moreover in another study (17) a microarraywas performed to identify the response to oxidative stress and neitherperR nor any of the genes identified in the PerR study (33) werede-repressed and we do not see any regulatory effect of manganese on theexpression of tdfl and znuA.

Previously, tdfl expression was reported to be induced in the presenceof active complement (18). In this microarray study expression profileswere compared of N. meningitidis grown in the presence of serum andheat-inactivated serum, and Tdfl was found 23-fold de-repressed in thepresence of the untreated serum. The relationship between zinc andcomplement regulation may not be obvious at first sight. A possibleexplanation for finding similar regulatory circuits may be that thebacteria in the array study were pre-grown in RMPI with BSA. Albumin isknown to chelate zinc, and therefore, pre-growth conditions may havebeen severely zinc-limited. Heat-treatment of human serum will releasezinc from albumin, thereby repressing tdfl expression. This explanationis strengthened by the fact that Tdfl expression is induced when BSA isadded to TSB medium during bacterial growth (data not shown).

A study by Hagen and Cornelissen (35) investigated whether any of theTdf proteins is essential for intracellular survival of N. gonorrhoeaein human epithelial cells. The authors also tested a Tdfl homologueknockout (NG1205), but this mutant was not affected in the intracellularsurvival.

The conservation of Tdfl is striking; with an identity of 98.6% amongthe sequenced N. meningitidis strains and a 99.2% similarity at theamino acid level of the mature protein. The Tdfl protein was found inall meningococci tested and all strains showed zinc-regulated expressionof tdfl. Between the Tdfl proteins of the sequenced meningococcal andgonococcal strains there is 96.1% identity and 97.3% similarity at theamino acid level. The differences between the sequences of Tdfl arescattered throughout the protein and do not cluster in a specific loop.We find an average 41% amino acid identity of Tdfl with homologs inother bacteria and in all cases the His/Asp stretch is conserved.Intriguingly, Tdfl homologs were particularly found in bacterial speciesresiding in the respiratory tract of humans and animals. Possibly in themucosal layers of the respiratory tract the unbound zinc concentrationis too low to allow sufficient passive diffusion through the porins andtherefore Tdfl becomes essential for bacterial growth and survival.While Tdfl is not essential for intracellular survival (35) it could beessential in the bodily fluids like serum and liquor where the free zincconcentration could also be very low. Also, we cannot rule out that Tdfladditionally recognizes a complexed form of zinc which may available inthe respiratory tract, serum and or cerebral fluid.

We have further shown that Tdfl can induce bactericidal antibodies inmice and that these antibodies are specifically directed at Tdfl. Alsowhen we used bacteria expressing Tdfl from the chromosomal locus wecould detect bactericidal activity, showing that during infection theantigen concentration is high enough to allow clearing of N.meningitidis.

The high level of conservation and the possibility to raiseTdfl-specific bactericidal antibodies make Tdfl an excellent vaccinecandidate.

Materials and Methods

Abbreviations used: IPTG, isopropyl β-D-1-thiogalactopyranoside, PAR,4-(2-pyridylazo)resorcinol, RPMI, Roswell Park Memorial Institute medium1640; Tdf, TonB-dependent family; TPEN,N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine, TSB, tryptic soybroth; ICP-MS, Inductively coupled plasma mass spectrometry.

Bacterial Strains and Growth Conditions.

Neisserial strains, listed in FIG. 5 are from the laboratory collection.Except when indicated otherwise, experiments were performed with strainHB-1 and mutants thereof. HB-1 is a non-encapsulated derivative ofserogroup B strain H44/76 (Bos & Tommassen, 2005). N. meningitidis wasgrown on GC agar (Oxoid) plates containing Vitox (Oxoid) and antibioticswhen appropriate (kanamycin, 100 μg/ml, chloramphenicol, 10 μg/ml) incandle jars at 37° C. Liquid cultures were grown in TSB (Difco) or inRPMI (Sigma) in plastic flasks at 37° C. with shaking. IPTG, zinc, andTPEN were added in the concentrations indicated s. Metals were added asa cocktail (340 nM ZnSO₄, 160 nM Na₂MoO₄, 800 nM MnCl₂, 80 nM CoCl₂ and80 nM CuSO₄ final concentrations) or as single compounds in the sameconcentrations as in the cocktail unless indicated otherwise. Ferricchloride was added as a final concentration of 8 μM. E. coli strainsDH5α and TOP10F′ (Invitrogen) were used for routine cloning andBL21(DE3) (Invitrogen) for expression. An E. coli hemA mutant was usedto assess the heme transport of Tdfl ((22). E. coli was propagated onLuria-Bertani medium supplemented when appropriate with 100 μg/mlampicillin, 50 μg/ml kanamycin, or 25 μg/ml chloramphenicol. For the E.coli heme-auxotroph C600 hemA::kan (22) the medium was supplemented with5-aminolevulinic acid.

Construction of Plasmids and Mutants.

All primers were designed on the MC58 genome sequence, using NMB0964(tdfl), NMB1730 (tonB), NMN0586 (znuA), NMB1266 (zur).

For high-level protein production in E. coli the tdfl gene without thesignal sequence-encoding part was amplified from chromosomal DNA ofstrain H44/76 by PCR using the primers0964-F-GATCATATGCATGAAACTGAGCAATCGGTG- (SEQ NO:4) and0964-R-GATGGATCCTTAAATCTTCACGTTCACGCCGCC- (SEQ NO:5) that carry therestriction sites NdeI and BamHI, respectively (bold). The resultingproduct was cloned into pCRII-TOPO according to the manufacturer'srecommendation (Invitrogen), yielding pCRII-tdfl, and subcloned intopET11a (Novagen) using NdeI/BamHI restriction, resulting in plasmidpET11a-tdfl.

To obtain a tdfl deletion construct, a kanamycin-resistance genecassette (36) was amplified by PCR with the primersKan-R-TGACGCGTCTCGACGCTGAGGTCTGC- (SEQ NO:6) andKan-F-TGTGTACAGTCGACTTCAGACGGCCACG- (SEQ NO:7) and cloned after MluI andBsrGI digestion into pCRII-tdfl digested with the same enzymes. In theresulting construct, pCRII-tdfl::kan, the kanamycin-resistance cassettesubstitutes for the region between by 437 and 1344 of tdfl.pCRII-tdfl::kan was used in a PCR with the 0964-R and 0964-F primers andthe resulting product was used to transform HB-1 (37).Kanamycin-resistant colonies were tested for correct gene replacement byPCR.

The entire tdfl gene from H44/76 was amplified with primersTdfl-F-GCATCATATGGCACAAACTACACTCAAACCC- (SEQ NO:8) andTdfl-R-ATGACGTCTTAAAACTTCACGTTCACGCCGCC- (SEQ NO:9) that containrecognition sites for NdeI and AatII (bold), respectively. The resultingPCR product was cloned into pCRII-TOPO and subcloned into pEN11-pldA(36) using NdeI and AatII restriction sites. The resulting plasmid,pEN11-tdfl, constitutes a Neisseria) replicative plasmid, containing alacI^(Q) gene and a tandem lac/tac promoter for controlled expression oftdfl.

The construct to generate a tonB knockout was made by amplifying DNAfragments upstream and downstream of the tonB gene using primers tonB-1(GTACGATGATTGTGCCGACC) (SEQ NO:10), tonB-2(ACTTTAAACTCCGTCGACGCAAGTCGACTGCGGGGGTTAA) (SEQ NO:11) with AccIrestriction sites (bold) for one fragment, and, tonB-3(TTAACCCCCGCAGTCGACTTGCGTCGACGGAGTTTAAAGT) (SEQ NO:12) with restrictionsite AccI (bold) and tonB-4 (GCCATACTGTTGCGGATTTGA) (SEQ NO:13) for theother fragment. The two fragments were each cloned into pCRII-TOPO andthen ligated to each other using the introduced restriction site AccIand the SpeI site in the pCRII-TOPO vector. The AccI site wassubsequently used to clone the chloramphenicol transacetylase gene frompKD3 (38) previously cloned into pCRII-TOPO by PCR amplification withprimers containing an AccI site. The resulting construct was amplifiedby PCR using primers tonB-1 and tonB-4 and this linear fragment was usedto transform N. meningitidis HB-1.

The zur gene was knocked out following the same strategy. Upstream anddownstream fragments were amplified in this case with primers: zur-1(TTCGCCGATGGCGGAATACA) (SEQ NO:14), zur-2(CTTTCAGCGCAAAGTCGACTCCGTCGACGCGTGCCTGTTC) (SEQ NO:15) with therestriction site AccI in bold, zur-3(GAACAGGCACGCGTCGACGGAGTCGACTTTGCGCTGAAAG) (SEQ NO:16) with therestriction site AccI in bold and zur-4 (TCCTATTGCGCAATACCCCC) (SEQNO:17).

A porA derivative of N. meningiditis strain H44/76, called CE2001 (39)was transformed with pMF121, resulting in deletion of the entire capsulelocus and production of lipopolysaccharide with a truncated outer core(36). A pLAFR-derived plasmid containing the tonB, exbB and exbD genesof N. meningitidis ((13) was described previously.

SDS-PAGE and Western Blot Analysis.

Cell lysates were prepared from bacteria grown for 6 hours. The cellswere diluted to OD_(600nm), 1, pelleted, and boiled in 100 μl SDS-PAGEsample buffer containing 2% SDS and 5% 2-mercaptoethanol. Proteins wereseparated by standard SDS-PAGE. Gels were either stained with Coomassiebrilliant blue or the proteins were transferred to nitrocellulosemembranes (Protran) using a wet transfer system (Biorad) in 25 mMTris-HCl, 192 mM glycine, 20% methanol. Membranes were blocked for 1 hin PBS containing 0.1% TWEEN® 20 and 0.5% PROTIFAR® (Nutricia). Blotswere incubated with antibodies in blocking buffer. Antibody binding wasdetected by using goat anti-rabbit IgG peroxidase-conjugated secondaryantibodies (Biosource) and enhanced chemiluminescence detection(Pierce).

Immunizations.

BL21(DE3) cells containing pET11a-tdfl were grown in LB to an OD A₆₀₀ of0.6 after which 1 mM IPTG was added and growth was continued for 2 h.The Tdfl protein accumulated in inclusion bodies, which were isolated asdescribed (40), and the purified protein was used to immunize rabbits atEurogentec. The resulting antiserum, SN1042, was used in a 1/5000dilution.

Outer membrane vesicles of strain CE1523/pEN11-tdfl grown in thepresence or absence of 1 mM IPTG, were prepared by deoxycholateextraction (41) and used to immunize mice as described (32). Sera fromten mice per group were collected after 42 days and pooled. Theexperiments complied with the relevant national guidelines of Belgiumand institutional policies of GlaxoSmithKline Biologicals.

RT-qPCR.

RT-qPCR was performed using an Applied Biosystems 7900HT Fast Real-TimePCR System and SYBR® green master mix (Applied Biosystems) according tothe manufacturer's recommendations. Total RNA was isolated byresuspending approximately 4×10⁹ Neisseria cells in 3 ml TRIZOL®(Invitrogen). After the addition of 600 μl chloroform andcentrifugation, the upper phase was mixed 1:1 with 75% ethanol. This wasloaded on a NUCLEOSPIN® RNA II column (Macherey-Nagel), which wassubsequently washed with buffer R3 from the NUCLEOSPIN® RNA II kit andeluted with 100 μl water. The RNA was then treated with TURBO DNA-FREE™(Ambion) to yield DNA-free RNA. To generate the cDNA, 1 μg of total RNAwas reverse transcribed from random hexamers using transcriptor Highfidelity cDNA synthesis kit (Roche) according to the manufacturer'srecommendations. As a control, parallel samples were prepared in whichthe reverse transcriptase was omitted from the reaction mixture. PCRswere performed in triplicate in a 25-μl volume in a 96-well plate(Applied Biosystems) with the following cycle parameters: 95° C. for 10min for enzyme activation followed by 40 cycles of 95° C. for 15 s and60° C. for 1 min. A melting plot was performed to ensure that the signaloriginated from the specific amplicon. Data analysis was performed usingthe comparative cycle threshold method (Applied Biosystems) to determinerelative expression levels. The rmpM transcript was used to normalizeall data.

ICP-MS.

Total zinc concentrations were measured by ICP-MS at the integratedlaboratory of the department of Geochemistry at the Utrecht University.N. meningitidis strains were grown in RPMI medium from a 0.1 starting ODA₅₅₀ for 6 h; at this time point a sample was taken and the remainingculture was grown for an additional hour in the presence of 1 μM zinc.After this hour, a second sample was taken. Both samples (7 ml) werewashed in phosphate-buffered saline and resuspended in water, killed for1 h at 56° C. and frozen at −80° C. The samples were then thawed,sonicated and filtered through 0.22-μm filters (Millipore).

PAR Competition Assay.

The PAR competition assay is a colorimetric reaction where the orangecolor of the PAR-zinc complex changes towards yellow in the presence ofa protein or chemical that is able to release zinc from PAR. The assaywas performed as described (42) with the following modifications:Instead of 50 μM we added 30 μM zinc and we first measured the PAR-zincsolution and then added the outer membrane vesicles to the cuvette andre-measured the solution. In this way we avoided the potential colorchange induced in time by UV. The data was then first normalized to thePAR-zinc measurement and then to the PAR alone sample to obtain thebinding values for the outer membrane vesicles. The results shown arethe normalized data of the absorption at 500 nm.

Serum Bactericidal Assay.

Wild-type H44/76 was transformed with pEN11-tdfl and inoculated fromovernight grown plates in TSB with 125 μM FeCl₃ with or without 1 mMIPTG in shaking flasks for 3 h at 37° C. until an OD A₅₅₀ of 0.5 wasreached. Serum to be tested was diluted 1:100 in Hank's balanced saltsolution (HBSS) (GIBCO), 0.3% BSA and then serially diluted (two-folddilution steps, eight dilutions) in a 50-μl volume in sterile U-bottom96-well microtiter plates (NUNC). Bacteria were diluted in HBSS, 0.3%BSA to yield ˜13,000 CFU per ml and 37.5 μl samples of the suspensionwere added to the serum dilutions. The microtiter plates were incubatedat 37° C. for 15 min while shaking. Subsequently, 12.5 μl of baby-rabbitcomplement (Pelfreez) or, as control for toxicity of the sera,heat-inactivated (56° C. for 45 min) complement was added to the wells.After 1 h incubation at 37° C. while shaking, the microtiter plates wereplaced on ice to stop the killing. Of each well, 20 μl was spotted on GCplates while plates were tilted to allow the drop to “run” down theplate. After overnight incubation, colonies were counted and thepercentage of killing was calculated. The bactericidal titer was definedas the highest serum dilution yielding >50% killing.

REFERENCES

-   1. Ratledge, C. 2007. Iron metabolism and infection. Food. Nutr.    Bull. 28:S515-523.-   2. Wandersman, C., and P. Delepelaire. 2004. Bacterial iron sources:    from siderophores to hemophores. Annu. Rev. Microbiol. 58:611-647.-   3. Wiener, M. C. 2005. TonB-dependent outer membrane transport:    going for Baroque? Curr. Opin. Struct. Biol. 15:394-400.-   4. Postle, K. 1993. TonB protein and energy transduction between    membranes. J. Bioenerg. Biomembr. 25:591-601.-   5. Braun, V. 2006. Energy transfer between biological membranes. ACS    Chem. Biol. 1:352-354.-   6. De, S. K., M. T. McMaster, and G. K. Andrews. 1990. Endotoxin    induction of murine metallothionein gene expression. J. Biol. Chem.    265:15267-15274.-   7. Corbin, B. D., E. H. Seeley, A. Raab, J. Feldmann, M. R.    Miller, V. J. Torres, K. L. Anderson, B. M. Dattilo, P. M.    Dunman, R. Gerads, R. M. Caprioli, W. Nacken, W. J. Chazin,    and E. P. Skaar. 2008. Metal chelation and inhibition of bacterial    growth in tissue abscesses. Science. 319:962-965.-   8. Stephens, D. S., and S. M. Zimmer. 2002. Pathogenesis, therapy,    and prevention of meningococcal sepsis. Curr. Infect. Dis. Rep.    4:377-386.-   9. Finne, J., M. Leinonen, and P. H. Mäkelä. 1983. Antigenic    similarities between brain components and bacteria causing    meningitis. Implications for vaccine development and pathogenesis.    Lancet. 2:355-357.-   10. Pettersson, A., A. Maas, and J. Tommassen. 1994. Identification    of the iroA gene product of Neisseria meningitidis as a lactoferrin    receptor. J. Bacteriol. 176:1764-1766.-   11. Legrain, M., V. Mazarin, S. W. Irwin, B. Bouchon, M. J.    Quentin-Millet, E. Jacobs, and A. B. Schryvers. 1993. Cloning and    characterization of Neisseria meningitidis genes encoding the    transferrin-binding proteins Tbp1 and Tbp2. Gene. 130:73-80.-   12. Lewis, L. A., E. Gray, Y. P. Wang, B. A. Roe, and D. W.    Dyer. 1997. Molecular characterization of hpuAB, the    haemoglobin-haptoglobin-utilization operon of Neisseria    meningitidis. Mol. Microbiol. 23:737-749.-   13. Stojiljkovic, I., V. Hwa, L. de Saint Martin, P. O'Gaora, X.    Nassif, F. Heffron, and M. So. 1995. The Neisseria meningitidis    haemoglobin receptor: its role in iron utilization and virulence.    Mol. Microbiol. 15:531-541.-   14. Carson, S. D., P. E. Klebba, S. M. Newton, and P. F.    Sparling. 1999. Ferric enterobactin binding and utilization by    Neisseria gonorrhoeae. J. Bacteriol. 181:2895-2901.-   15. Turner, P. C., C. E. Thomas, I. Stojiljkovic, C. Elkins, G.    Kizel, D. A. Ala'Aldeen, and P. F. Sparling. 2001. Neisserial    TonB-dependent outer-membrane proteins: detection, regulation and    distribution of three putative candidates identified from the genome    sequences. Microbiology. 147:1277-1290.-   16. Ducey, T. F., M. B. Carson, J. Orvis, A. P. Stintzi, and D. W.    Dyer. 2005. Identification of the iron-responsive genes of Neisseria    gonorrhoeae by microarray analysis in defined medium. J. Bacteriol.    187:4865-4874.-   17. Grifantini, R., E. Frigimelica, I. Delany, E. Bartolini, S.    Giovinazzi, S. Balloni, S. Agarwal, G. Galli, C. Genco, and G.    Grandi. 2004. Characterization of a novel Neisseria meningitidis Fur    and iron-regulated operon required for protection from oxidative    stress: utility of DNA microarray in the assignment of the    biological role of hypothetical genes. Mol. Microbiol. 54:962-979.-   18. Dove, J. E., K. Yasukawa, C. R. Tinsley, and X. Nassif. 2003.    Production of the signalling molecule, autoinducer-2, by Neisseria    meningitidis: lack of evidence for a concerted transcriptional    response. Microbiology. 149:1859-1869.-   19. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z.    Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and    PSI-BLAST: a new generation of protein database search programs.    Nucleic Acids Res. 25:3389-3402.-   20. Furano, K., and A. A. Campagnari. 2004. Identification of a    hemin utilization protein of Moraxella catarrhalis (HumA). Infect.    Immun. 72:6426-6432.-   21. Mazoy, R., and M. L. Lemos. 1996. Identification of heme-binding    proteins in the cell membranes of Vibrio anguillarum. FEMS    Microbiol. Lett. 135:265-270.-   22. Ghigo, J. M., S. Létoffé, and C. Wandersman. 1997. A new type of    hemophore-dependent heme acquisition system of Serratia marcescens    reconstituted in Escherichia coli. J. Bacteriol. 179:3572-3579.-   23. Fetzer, S. I., and K. Hantke. 1998. The ZnuABC high-affinity    zinc uptake system and its regulator Zur in Escherichia coli. Mol.    Microbiol. 28:1199-1210.-   24. Ferguson, A. D., E. Hofmann, J. W. Coulton, K. Diederichs,    and W. Welte. 1998. Siderophore-mediated iron transport: crystal    structure of FhuA with bound lipopolysaccharide. Science.    282:2215-2220.-   25. Yatsunyk, L. A., J. A. Easton, L. R. Kim, S. A. Sugarbaker, B.    Bennett, R. M. Breece, Vorontsov, I I, D. L. Tierney, M. W. Crowder,    and A. C. Rosenzweig. 2008. Structure and metal binding properties    of ZnuA, a periplasmic zinc transporter from Escherichia coli. J.    Biol. Inorg. Chem. 13:271-288.-   26. Bentley, S. D., G. S. Vernikos, L. A. Snyder, C. Churcher, C.    Arrowsmith, T. Chillingworth, A. Cronin, P. H. Davis, N. E.    Holroyd, K. Jagels, M. Maddison, S. Moule, E. Rabbinowitsch, S.    Sharp, L. Unwin, S. Whitehead, M. A. Quail, M. Achtman, B.    Barrell, N. J. Saunders, and J. Parkhill. 2007. Meningococcal    genetic variation mechanisms viewed through comparative analysis of    serogroup C strain FAM18. PLoS Genet. 3:e23.-   27. Dempsey, J. A., W. Litaker, A. Madhure, T. L. Snodgrass,    and J. G. Cannon. 1991. Physical map of the chromosome of Neisseria    gonorrhoeae FA1090 with locations of genetic markers, including opa    and pil genes. J. Bacteriol. 173:5476-5486.-   28. Parkhill, J., M. Achtman, K. D. James, S. D. Bentley, C.    Churcher, S. R. Klee, G. Morelli, D. Basham, D. Brown, T.    Chillingworth, R. M. Davies, P. Davis, K. Devlin, T. Feltwell, N.    Hamlin, S. Holroyd, K. Jagels, S. Leather, S. Moule, K.    Mungall, M. A. Quail, M. A. Rajandream, K. M. Rutherford, M.    Simmonds, J. Skelton, S. Whitehead, B. G. Spratt, and B. G.    Barrell. 2000. Complete DNA sequence of a serogroup A strain of    Neisseria meningitidis Z2491. Nature. 404:502-506.-   29. Tettelin, H., N. J. Saunders, J. Heidelberg, A. C.    Jeffries, K. E. Nelson, J. A. Eisen, K. A. Ketchum, D. W.    Hood, J. F. Peden, R. J. Dodson, W. C. Nelson, M. L. Gwinn, R.    DeBoy, J. D. Peterson, E. K. Hickey, D. H. Haft, S. L. Salzberg, O.    White, R. D. Fleischmann, B. A. Dougherty, T. Mason, A.    Ciecko, D. S. Parksey, E. Blair, H. Cittone, E. B. Clark, M. D.    Cotton, T. R. Utterback, H. Khouri, H. Qin, J. Vamathevan, J.    Gill, V. Scarlato, V. Masignani, M. Pizza, G. Grandi, L. Sun, H. O.    Smith, C. M. Fraser, E. R. Moxon, R. Rappuoli, and J. C.    Venter. 2000. Complete genome sequence of Neisseria meningitidis    serogroup B strain MC58. Science. 287:1809-1815.-   30. Chen, C. Y., and S. A. Morse. 2001. Identification and    characterization of a high-affinity zinc uptake system in Neisseria    gonorrhoeae. FEMS Microbiol. Lett. 202:67-71.-   31. Ammendola, S., P. Pasquali, C. Pistoia, P. Petrucci, P.    Petrarca, G. Rotilio, and A. Battistoni. 2007. The high affinity    Zn²⁺ uptake system ZnuABC is required for bacterial zinc homeostasis    in intracellular environments and contributes to virulence of    Salmonella enterica. Infect. Immun. 75:5867-5876.-   32. Stewart, A. J., C. A. Blindauer, S. Berezenko, D. Sleep,    and P. J. Sadler. 2003. Interdomain zinc site on human albumin.    Proc. Natl. Acad. Sci. USA. 100:3701-3706.-   33. Wu, H. J., K. L. Seib, Y. N. Srikhanta, S. P. Kidd, J. L.    Edwards, T. L. Maguire, S. M. Grimmond, M. A. Apicella, A. G.    McEwan, and M. P. Jennings. 2006. PerR controls Mn-dependent    resistance to oxidative stress in Neisseria gonorrhoeae. Mol.    Microbiol. 60:401-416.-   34. Nanamiya, H., G. Akanuma, Y. Natori, R. Murayama, S. Kosono, T.    Kudo, K. Kobayashi, N. Ogasawara, S. M. Park, K. Ochi, and F.    Kawamura. 2004. Zinc is a key factor in controlling alternation of    two types of L31 protein in the Bacillus subtilis ribosome. Mol.    Microbiol. 52:273-283.-   35. Hagen, T. A., and C. N. Cornelissen. 2006. Neisseria gonorrhoeae    requires expression of TonB and the putative transporter TdfF to    replicate within cervical epithelial cells. Mol. Microbiol.    62:1144-1157.-   36. Bos, M. P., B. Tefsen, P. Voet, V. Weynants, J. P. M. van    Putten, and J. Tommassen. 2005. Function of neisserial outer    membrane phospholipase A in autolysis and assessment of its vaccine    potential. Infect. Immun. 73:2222-2231.-   37. Voulhoux, R., M. P. Bos, J. Geurtsen, M. Mols, and J.    Tommassen. 2003. Role of a highly conserved bacterial protein in    outer membrane protein assembly. Science. 299:262-265.-   38. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation    of chromosomal genes in Escherichia coli K-12 using PCR products.    Proc. Natl. Acad. Sci. USA. 97:6640-6645.-   39. Tommassen, J., P. Vermeij, M. Struyvé, R. Benz, and J. T.    Poolman. 1990. Isolation of Neisseria meningitidis mutants deficient    in class 1 (PorA) and class 3 (PorB) outer membrane proteins.    Infect. Immun. 58:1355-1359.-   40. Dekker, N., K. Merck, J. Tommassen, and H. M. Verheij. 1995. In    vitro folding of Escherichia coli outer-membrane phospholipase A.    Eur. J. Biochem. 232:214-219.-   41. Weynants, V. E., C. M. Feron, K. K. Goraj, M. P. Bos, P. A.    Denoel, V. G. Verlant, J. Tommassen, I. R. Peak, R. C. Judd, M. P.    Jennings, and J. T. Poolman. 2007. Additive and synergistic    bactericidal activity of antibodies directed against minor outer    membrane proteins of Neisseria meningitidis. Infect. Immun.    75:5434-5442.-   42. Lim, K. H., C. E. Jones, R. N. vanden Hoven, J. L.    Edwards, M. L. Falsetta, M. A. Apicella, M. P. Jennings, and A. G.    McEwan. 2008. Metal binding specificity of the MntABC permease of    Neisseria gonorrhoeae and its influence on bacterial growth and    interaction with cervical epithelial cells. Infect. Immun.    76:3569-3576.

TABLE 1 Conservation of the mature Tdfl protein sequence in thesequenced Neisseria strains. Identity (%) Strain MC58 Fam18 Z2491 053442FA1090 NCCP11945 ST-640 Similarity (%) N. meningitidis 730/734 720/734720/734 706/734 707/734 712/734 MC58 (99.5) (98.1) (98.1) (96.2) (96.3)(97.0) N. meningitidis 733/734 722/734 718/734 705/734 706/734 712/734Fam18 (99.9) (98.4) (97.8) (96.0) (96.2) (97.0) N. meningitidis 725/734726/734 716/734 707/734 706/734 710/734 Z2491 (98.8) (98.9) (97.5)(96.3) (96.2) (96.7) N. meningitidis 726/734 727/734 723/734 706/734707/734 707/734 053442 (98.9) (99.0) (98.5) (96.2) (96.3) (96.3) N.gonorrhoeae 715/734 714/734 714/734 715/734 733/734 702/734 FA1090(97.4) (97.3) (97.3) (97.4) (99.9) (95.6) N. gonorrhoeae 716/734 715/734713/734 716/734 733/734 701/734 NCCP11945 (97.5) (97.4) (97.1) (97.5)(99.9) (95.5) N. lactamica 717/734 718/734 718/734 715/734 711/734710/734 ST-640 (97.7) (97.8) (97.8) (97.4) (96.9) (96.7)

1. An immunogenic composition comprising: (a) a Zn2+ salt; (b) apharmaceutically acceptable excipient; and (c) a polypeptide selectedfrom the group consisting of: (i) a polypeptide comprising an amino acidsequence having 95% identity to the amino acid sequenceRDQYGLPAHSHEYDDCHADIIWQKSLINKRYLQLYPHLLTEEDIDYDNPGLSCGFHDDDNAHAHT HS(SEQ NO: 18), and (ii) a polypeptide comprising an immunogenic fragmentof 20 or more contiguous amino acids of the amino acid sequenceRDQYGLPAHSHEYDDCHADIIWQKSLINKRYLQLYPHLLTEEDIDYDNPGLSCGFHDDDNAHAHT HS(SEQ NO: 18).
 2. The immunogenic composition of claim 1 wherein thepolypeptide comprises the amino acid sequenceRDQYGLPAHSHEYDDCHADIIWQKSLINKRYLQLYPHLLTEEDIDYDNPGLSCGFHDDDNAHAHT HS(SEQ NO: 18).
 3. A method of eliciting an immune response againstNeisseria, said method comprising the steps of: administering to amammal an immunologically effective amount of an immunogenic compositionof claim
 1. 4. A method of eliciting an immune response againstNeisseria, said method comprising the steps of: administering to amammal an immunologically effective amount of an immunogenic compositionof claim 2.